WO2021222178A1

WO2021222178A1 - Adjunct therapy for suppressing immune response against gene therapy

Info

Publication number: WO2021222178A1
Application number: PCT/US2021/029304
Authority: WO
Inventors: Roland Herzog; John Butterfield
Original assignee: The Trustees Of Indiana University
Priority date: 2020-04-27
Filing date: 2021-04-27
Publication date: 2021-11-04

Abstract

Gene therapy for patients with hemophilia or other inherited diseases is often complicated by deleterious immune responses, which prevent desired gene therapy outcomes. A therapeutic protocol as an adjunct to gene therapy modulates the immune response by co-administration of an immunosuppressive and an agent effective to suppress the interleukin- 15 receptor.

Description

ADJUNCT THERAPY FOR SUPPRESSING IMMUNE RESPONSE AGAINST GENE THERAPY

[0001] REFERENCE TO GOVERNMENT GRANTS

[0002] This invention was made with government support under HL131093 awarded by National Institutes of Health. The Government has certain rights in the invention.

[0003] FIELD OF THE INVENTION

[0004] The general field of the present disclosure is gene therapy for patients with hemophilia or other inherited diseases. More particularly, the present disclosure addresses the problem that gene therapy often is complicated by cytotoxic immune responses, which prevent desired gene therapy outcomes.

[0005] BACKGROUND

[0006] Adeno-associated virus (AAV) was first discovered from laboratory adenovirus preparations in the mid-1960s and found in human tissues soon after. Several important aspects of AAV were characterized, including its genome configuration and composition, DNA replication and transcription, infectious latency and virion assembly. Subsequently, investigators successfully cloned the wild-type AAV2 sequence into plasmids, which enabled genetic studies and sequencing of the entire AAV2 genome. These early investigations provided fundamental knowledge that led to the use of AAV as a gene delivery vehicle. Since the advent of AAV vectors, their use as a biotherapy has also advanced the understanding of virus-host interactions that govern the transduction pathway of AAV.

[0007] Today, recombinant AAVs are the leading platform for in vivo delivery of gene therapies. The first recombinant AAV gene therapy product, alipogene tiparvovec (GLYBERA®), was approved by the European Medicines Agency to treat lipoprotein lipase deficiency in 2012, while the approval of voretigene neparvovec-rzyl (LUXTURNA®), the first recombinant AAV gene therapy product licensed in the United States, followed 5 years later. Although the clinical success of recombinant AAV gene therapy is encouraging, several limitations and challenges of this gene delivery platform persist, including, for example, issues with recombinant AAV manufacturing and more importantly, immunological barriers to delivery.

[0008] Most recombinant AAV gene therapy research has focused on the liver, striated muscles and the CNS. Almost all natural AAV capsids can transduce liver efficiently following systemic administration. Thus, recombinant AAVs provide a robust liver-targeting platform to treat a variety of diseases such as hemophilia A and hemophilia B, familial hypercholesterolemia, ornithine transcarbamylase deficiency and Crigler-Najjar syndrome. Certain capsids can target multiple muscle types throughout the body, enabling recombinant AAV gene therapies to be developed for multiple muscle diseases, especially those afflicting muscles of the entire body, such as Duchenne muscular dystrophy and the like. In addition, transduced muscle can serve as a bio factory to produce secreted therapeutic proteins for the treatment of non-muscle diseases. Several genes involved in signaling and metabolism have been tested to treat heart failure.

[0009] Recombinant AAV gene therapy focused on the CNS, including the brain and eye, is also under clinical development. As noted, the first recombinant AAV gene therapy drug approved by the US Food and Drug Administration (FDA), voretigene neparvovec-rzyl (LUXTURNA®), treats patients with an inherited form of vision loss caused by RPE65 gene mutations. Regarding the brain, some investigators have found that direct intraparenchymal recombinant AAV injections result in localized distribution of recombinant AAV and are ideal for the treatment of CNS diseases that afflict a defined region of the brain, such as the putamen in Parkinson disease. Delivery to the cerebrospinal fluid space by intrathecal injection, on the other hand, can achieve broader CNS distribution. Alternatively, intravenous delivery of certain serotype vectors, such as AAV9 and AAVrh.10, has allowed the vectors to cross the blood brain barrier to transduce neurons and glia. Thus, systemic recombinant AAV administration can be used to target diseases that afflict widespread regions of the CNS, including spinal muscular atrophy, amyotrophic lateral sclerosis, Canavan disease, GM1 gangliosidosis and mucopolysaccharidosis type III.

[0010] Hemophilia is a disease of humans and other mammals wherein a gene encoding a blood coagulation factor contains a mutation such that the encoded protein does not function normally in the cascade process. According to a recent report of the World Federation of Hemophilia (WFH), there are 28,775 patients worldwide with hemophilia B and 143,523 patients who have hemophilia A. There are around 20,000 hemophilia patients in United States with hemophilia A being about 6 times more common than hemophilia B. The promising outcome of liver-directed gene transfer in both hemophilia A and B patients provides a great deal of hope in curing hemophilic patients with a single treatment. However, although hepatic gene transfer has the potential to induce immune tolerance, adjunct immune modulation may further enforce tolerance to the factor VIII (FVIII) or FIX transgene products.

[0011] Clinical studies have shown that immune responses to AAV vectors and their transgene products represent one of the greatest hurdles to the success of gene therapy. These immune responses encompass both cellular responses against AAV transduced cells and humoral immune responses against the AAV capsid. It took investigators by surprise when two patients in AAV mediated clinical trial of hemophilia B developed cellular immunogenicity against the viral capsid that led to a sudden decline in factor IX (FIX) expression associated with asymptomatic transient transaminitis (Manno, C.S. et al. Successful transduction of liver in hemophilia by AAV -Factor IX and limitations imposed by the host immune response. Nature Med. 12, 342-347 (2006)).

[0012] Similarly, CD8⁺ T cell responses against the transgene product were observed in patients receiving AAV gene therapy for muscular dystrophy or al -antitrypsin deficiency (Calcedo, R. et al. Class I-restricted T-cell responses to a polymorphic peptide in a gene therapy clinical trial for alpha- 1 -antitrypsin deficiency. PNAS 114, 1655-1659 (2017); Mendell, J.R. et al. Dystrophin immunity in Duchenne's muscular dystrophy. NEJM 363, 1429-1437 (2010)).

[0013] Though liver has been shown to induce immunological tolerance to transgene product, concerns remain that AAV encoded therapeutic protein might be recognized as non-self protein and would be a potential target of immune cells. Moreover, an inflammatory milieu due to innate sensing of AAV capsid or other concurrent immune responses could provide relay signal to activate anti-transgene immune response. Thus far, however, there is a scarcity of data on: i) immune responses to transgene in AAV mediated liver gene transfer, ii) innate sensors involved in transgene specific immunity, and, iii) underlying mechanisms of transgene-specific immune responses. Additionally, ongoing clinical studies involving liver gene transfer in hemophilia patients rely on general immune suppression to subdue immune responses to vector and/or transgene product. These immunosuppressive agents increase the risk of opportunistic infections along with their various side effects. Identifying the immune players and understanding the underlying mechanisms of these immune responses (to AAV-capsid and transgene product) would provide new potential targets and pathways that could specifically be blocked (contrary to general immune suppression) to prevent immune responses in the context of liver gene therapy.

[0014] Prior studies have implicated innate immune sensors such as Toll-like receptors (TLR) 2 and 9 and their downstream adaptor molecule myeloid differentiation primary response protein 88 (MyD88) in sensing viral capsid and genome and mediating adaptive immune response against AAV (Martino, A.T. et al. The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver. Blood 117, 6459-6468 (2011); Hosel, M. et al. Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors. Hepatology 55, 287-297 (2012); Sudres, M. et al. MyD88 signaling in B cells regulates the production of Thl -dependent antibodies to AAV. Molecular Therapy 20, 1571-1581 (2012); Rogers, G.L. et al. Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer. J. Innate Immunity 7, 302-314 (2015)).

[0015] The inventors have previously discovered that that co-administration of an interleukin- 1 (IL-1) signaling pathway disrupter such as an anti-IL antibody to a mammal with a gene delivery vehicle results in suppression of a cytotoxic CD8⁺ T cell response against the AAV vector or a transgene product expressed from the AAV vector. It was discovered that inhibiting an immune response in a subject undergoing AAV gene therapy was particularly useful for gene therapy vectors designed for the treatment of hemophilia.

[0016] However, it was further discovered that hepatic gene transfer of codon-optimized human factor VIII (FVIII) with an adeno-associated virus serotype 8 (AAV8-co/A) in hemophilia A animal models can induce tolerance and durably replace FVIII. However, in some hemophilia A animal models, tolerance is not always observed. For instance, it was observed that a sub-strain of BALB/c-HA that reliably forms FVIII function-inhibiting antibodies (inhibitors) with this same approach.

[0017] Formation of such FVIII function-inhibiting antibodies provides a significant impediment to AAV gene therapy in hemophilia.

[0018] Therefore, what is needed are methods to induce a transient immune suppression to prevent this response that does not impair the underlying AAV gene therapy.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A-C depicts the results showing that treatment with rapamycin or rapamycin + anti -IL- 15 (FIG. 1A) prevents antibody formation against human factor VIII (FVIII) in hepatic AAV8-hFVIII (AAV8-coF8) gene transfer to hemophilia A mice (FIG. IB). However, anti-IL-15 was required to prevent loss of FVIII expression after rapamycin was discontinued (FIG. 1C). [0021] FIG. 2A-C depicts the detection of antibodies against AAV 8 capsid 16 weeks after gene transfer in animals that had received treatment with either rapamycin or rapamycin + anti-IL-15 (FIG. 2A). All animals received injections of a second AAV8 vector (same dose as original vector, 2xl0expl 1/mouse, n=7-9 per experimental group), this time, however, they expressed human factor IX (AAV8-hFIX). When tested 3 weeks later, those mice that received treatment with either rapamycin or rapamycin + anti-IL-15 showed similar FIX expression as previously naive control mice, while mice that had initially been injected with AAV8-FVIII vector without immune suppression failed to express FIX (FIG. 2B). After this second vector administration, which was done without immune suppression, animals of all experimental groups formed antibodies to AAV8 capsid (FIG. 2C).

[0022] FIG. 3A-B depicts the sustained expression of FVIII activity in hemophilia A mice after hepatic AAV gene transfer combined with immune modulation using rapamycin and anti-IL-15. FIG. 3A shows 6-8 week-old-old BALB/c-HA mice received intravenous AAV8-hFVIII (2xlOⁿ vg). Control mice (n=10) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=8) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). FIG 3B shows FVIII activity as measured by chromogenic assay. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA at each time point, corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, pO.OOl; ****, pO.OOOl. [0023] FIG. 4A-D show the results following eight weeks of rapamycin with AAV gene transfer that prevents antibodies to FVIII and capsid, allowing AAV readministration. Six-eight week-old BALB/c-HA mice received intravenous AAV8-hFVIII (2xlOⁿ vg). Control mice (n=10) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=8) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). 17 weeks after initial gene transfer, naive BALB/c-HA mice (n=8) and all surviving mice previously administered AAV8-hFVIII, received AAV8-hFIX (2x10¹¹ vg). At 16 weeks after gene transfer, (FIG. 4A) FVIII inhibitor antibodies were measured by Bethesda assay and ELISA measured (FIG. 4B) a-AAV8 IgG2a antibodies. At 20 weeks after gene transfer, ELISA measured (FIG. 4C) circulating human clotting factor IX (hFIX) and (FIG. 4D) a- AAV 8 IgG2a antibodies. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA, corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, pO.OOl; ****, pO.OOOl.

[0024] FIG. 5A-B shows the results confirming that that anti-IL-15/rapamycin combination helps sustain FVIII activity in hemophilia A mice, while rapamycin treatment followed by CD8⁺ T cell depletion was less successful. (FIG. 5A) 6-8 week-old BALB/c-HA mice received intravenous AAV8-hFVIII (2x10¹¹ vg). Control mice (n=6) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=21) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). After 8 weeks rapamycin-treated mice (n=8/21) received anti-CD8 (4 mg/kg) intravenously 2x/week for 8 weeks. (FIG. 5B) FVIII activity was measured by chromogenic assay. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA at each time point and corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, pO.OOl; ****, pO.OOOl.

[0025] FIG. 6A-E shows the results of hFVIII gene copy numbers and mRNA transcripts are maintained regardless of FVIII activity, suggesting a translational shutdown that can be prevented by adding anti-IL-15 to rapamycin treatment. Six-eight week-old BALB/c-HA mice received either received no treatment (naive; n=7) or intravenous AAV8-hFVIII (2x1011 vg). Control mice (n=6) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=21) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). After 8 weeks, some rapamycin-treated mice received anti-CD8 (4 mg/kg; n=8) intravenously 2x/week for 8 weeks. At 16 weeks after gene transfer, (FIG. 6A) FVIII inhibitors were measured by Bethesda assay, (FIG. 6B) AAV copy number was measured by qPCR of DNA extracted from liver tissue, and (FIG. 6C) hFVIII and b-actin (reference gene) mRNA transcripts were measured by RT-qPCR of RNA extracted from liver tissue and fold change calculated by 2-AACt method. (FIG. 6D and 6E) A simple linear regression line was drawn comparing (FIG. 6D) AAV copy number and hFVIII mRNA and (FIG. 6E) hFVIII mRNA and FVIII activity. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA and corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, pO.OOOl.

[0026] FIG. 7A-E — CHANGE FROM COLOR shows that anti-IL-15 treatment protects AAV-FVIII transduced livers of hemophilia A mice from immune attack and preserves FVIII expression in hepatocytes. FIG. 7A-E are representative examples of liver sections with IHC staining for hFVIII (green), CD8 (red; white arrows), and nuclei (blue) are depicted. Six-eight week old BALB/c-HA mice received intravenous AAV8-hFVIII (2xlOⁿ vg). Control mice (n=6) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (4 mg/kg), or rapamycin/anti-IL-15 (4 mg/kg). After 8 weeks, some rapamycin-treated mice received anti-CD8 (4 mg/kg) intravenously 2x/week for 8 weeks. (FIG. 7A) naive mice or 16 weeks after gene transfer: (FIG. 7B) AAV only or with regimens of (FIG. 7C and FIG. 7D) rapamycin, (FIG. 7D) rapamycin/anti-CD8, or (FIG. 7E) rapamycin/anti-IL-15.

[0027] SUMMARY OF THE DISCLOSURE

[0028] The present disclosure provides methods for preserving AAV gene therapy through transient immune suppression, comprising co-administering to a subject undergoing AAV gene therapy an effective amount of an immunosuppressive agent and an interleukin- 15 (IL-15) blocker, the combination of an immunosuppressive agent and IL-15 blocker being effective to preserve therapeutic expression of a transgene delivered by said AAV gene therapy.

[0029] In other embodiments, co-administering an effective amount of rapamycin and an interleukin- 15 blocker is effective to suppress a deleterious immune response against the AAV gene therapy.

[0030] In other embodiments, co-administering an effective amount of an immunosuppressive agent and an interleukin- 15 blocker is effective to suppress a memory CD8⁺ T cell response. [0031] In any of the embodiments, the methods provide that the AAV vector is a liver-targeting AAV or AAV mediated liver gene therapy. In still other embodiments, the AAV vector is a liver gene transfer vector.

[0032] In any of the embodiments of the invention, the IL-15 blocker includes one or more of an antagonist of IL-15 receptors or antibody against IL-15 receptors. In any of the embodiments, the IL-15 blocker is one or more an agent effective to deactivate IL-15 receptors.

[0033] In any of the embodiments of the invention the IL-15 blocker is administered prior to, concomitant with or following administration of one or more immunosuppressive agents or compounds.

[0034] In still other embodiments, the immunosuppressive agent is an mTOR inhibitor. In any embodiment of the invention the mTOR inhibitor is selected from one or more of rapamycin, temsirolimus, everolimus, ridaforolimus, tacrolimus, or the like. In a preferred embodiment, the mTOR inhibitor is rapamycin.

[0035] In any of the embodiments of the invention, the co-administration of each of the mTOR inhibitor and the IL-15 blocker to the subject occurs within a suitable time prior to, during, or following gene therapy treatment. In some embodiments, the mTOR inhibitor and the IL-15 blocker are administered together. In yet other embodiments, mTOR inhibitor and the IL-15 blocker are administered separately.

[0036] The present invention provides a method of treating a mammal comprising: (a) administering a recombinant AAV virus vector to a mammal; and (b) co-administering each of an immunosuppressive agent and an IL-15 blocker to the mammal, each in an amount effective to have a therapeutic effect on said mammal.

[0037] In any of the embodiments of the current invention, the method elicits a therapeutic effect where the therapeutic effect is to induce a transient immune suppression to prevent an immune response that would impair the underlying AAV gene therapy.

[0038] In any of the embodiments of the invention, the mammal is a patient in need of gene therapy treatment.

[0039] In still other embodiments, administration of the immunosuppressive agent occurs orally and the IL-15 blocker is injected any one of intramuscularly, intraperitoneally or intravenously into the mammal. Yet other embodiments the immunosuppressive agent and the IL-15 blocker are administered any one or more of orally, intramuscularly, intraperitoneally or intravenously into the mammal.

[0040] In any embodiment, the IL-15 blocker is injected at a single site per dose or multiple sites per dose. [0041] In any embodiment of the current invention, all of the materials can be packaged into a kit containing all of the necessary components to carry out the claimed methods.

[0042] These and other embodiments and features of the disclosure will become more apparent through reference to the following description, the accompanying figures, and the claims. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and permutations.

[0043] DETAILED DESCRIPTION

[0044] Throughout this disclosure, various quantities, such as amounts, sizes, dimensions, proportions and the like, are presented in a range format. It should be understood that the description of a quantity in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as all individual numerical values within that range unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 4.62, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0045] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B, or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). [0046] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0047] Immunosuppressive agents such as the mTOR inhibitor, rapamycin, are increasingly used to prevent immune responses in gene therapy. These drugs block effector T cell and antibody responses and also promotes regulatory T cells. However, they also promote memory T cell responses. As a result, cessation of such administration of an immunosuppressive agent such as rapamycin may result in an immune response kicking in due to cessation of immune suppression. The present disclosure is based on the discovery that co-administration of rapamycin with an agent effective to suppress the functionality of interleukin- 15 (IL-15) receptors suppresses an immune response following cessation of rapamycin administration. The terms “suppress” or “suppression” or other formatives thereof, and also the terms “inhibit” or “inhibition” or other formatives thereof, when used herein to describe an immune response, are intended to refer to a reduction in or prevention of the immune response.

[0048] While methods for inhibiting an immune response in a subject undergoing AAV gene therapy disclosed herein are particularly useful for gene therapy vectors designed for the treatment of hemophilia, the disclosure is not limited solely to the treatment of hemophilia. Rather, the disclosure should be construed to include co-administration of an IL-15 receptor blocking agent and an immunosuppressive agent where the AAV gene therapy includes without limitation a variety of DNA encoding gene products that are useful for the treatment of other disease states in a mammal. In some embodiments, the gene therapy vector is a liver-targeting AAV, such as, for example, a liver gene transfer vector. In some embodiments, the AAV gene therapy comprises AAV mediated liver gene therapy. Alternative DNA incorporated into the AAV gene therapy vector and associated disease states include, but are not limited to: DNA encoding glucose-e- phosphatase, associated with glycogen storage deficiency type 1A; DNA encoding phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency; DNA encoding galactose- 1 phosphate uridyl transferase, associated with galactosemia; DNA encoding phenylalanine hydroxylase, associated with phenylketonuria; DNA encoding branched chain . alpha. -ketoacid dehydrogenase, associated with Maple syrup urine disease; DNA encoding fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; DNA encoding methylmalonyl-CoA mutase, associated with methylmalonic acidemia; DNA encoding medium chain acyl CoA dehydrogenase, associated with medium chain acetyl CoA deficiency; DNA encoding ornithine transcarbamylase, associated with ornithine transcarbamylase deficiency; DNA encoding argininosuccinic acid synthetase, associated with citrullinemia; DNA encoding low density lipoprotein receptor protein, associated with familial hypercholesterolemia; DNA encoding UDP-glucouronosyltransferase, associated with Crigler-Najjar disease; DNA encoding adenosine deaminase, associated with severe combined immunodeficiency disease; DNA encoding hypoxanthine guanine phosphoribosyl transferase, associated with Gout and Lesch-Nyan syndrome; DNA encoding biotinidase, associated with biotinidase deficiency; DNA encoding b-glucocerebrosidase, associated with Gaucher disease; DNA encoding 3 -glucuronidase, associated with Sly syndrome; DNA encoding peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; DNA encoding porphobilinogen deaminase, associated with acute intermittent porphyria; DNA encoding a-1 antitrypsin for treatment of a-1 antitrypsin deficiency (emphysema); DNA encoding erythropoietin for treatment of anemia due to thalassemia or to renal failure; and, DNA encoding insulin for treatment of diabetes.

[0049] Specifically, the types of diseases, tissues and AAV targets that are contemplated by the current disclosure include those shown in Table 1. It should be noted that Table 1 is a representation of non-limiting examples.

[0050] TABLE 1:

A1 AT, od-antitrypsin; AADC, aromatic 1-amino acid decarboxylase; AMD, age-related macular degeneration; ARSB, arylsulfatase B; CLN2, neuronal ceroid lipofuscinosis type 2; CMT1A, Charcot-Marie-Tootli disease type 1A; CNGB3, cyclic nucleotide-gated channel-P3; DMD, Duclienne muscular dystrophy; DYSF, dysferlin; FH, familial hypercliolesterolaemia; FVIII, factor VIII; G6PC, glucose-6-pliosphatase catalytic subunit; GAA, ot- glucosidase; GAN, gigaxonin; GDNF, glial cell line-derived neurotrophic factor; GSDla, glycogen storage disease type la; LCA, Leber congenital amaurosis; LDLR, low-density lipoprotein receptor; LHON, Leber hereditary optic neuropathy; MPS, mucopolysaccharidosis; MTM, myotubular myopathy; NAGLU, V-a-acetylglucosaminidase; NTF3, neurotrophin 3; OTC, ornithine transcarbamylase; REP1, RAB escort protein 1; RLBPl, retinaldehydebinding protein 1; RP, retinitis pigmentosa; RPE65, retinal pigment epithelium-specific 65 kDa protein; RPGR, retinitis pigmentosa GTPase regulator; RSI, retinoschisin 1; SGSH, TV-sulfoglucosamine sulfohydrolase; SMA, spinal muscular atrophy; SMN, survival of motor neuron; UGT1A1, UDP glucuronosyltransferase family 1 member At; VEGF, vascular endothelial growth factor; ZFN, zinc -finger-containing protein. (Adapted in part from: Wang et al., “Adeno-associated virus vector as a platform for gene therapy delivery,” Nature Reviews 18: 2019, pp. 358-378).

[0051] The methods of the disclosure can include without limitation, gene replacement therapies in which the ultimate goal is to deliver a gene product to compensate for loss-of-function mutations. Gene replacement is suitable for treating recessive monogenic diseases. A non-limiting example of gene replacement target is the treatment of hemophilia A or B.

[0052] In other embodiments, the methods of the disclosure can include without limitation, gene silencing where the therapeutic goal is to silence genes that produce toxic mutations. One nonlimiting example is Huntington disease.

[0053] In still other embodiments, the methods of the disclosure can include without limitation gene addition therapies. Such therapies can target complex genetic diseases and acquired diseases including but not limited to heart failure and infectious diseases. Gene addition can modulate these diseases in multiple ways, such as supplying neurotrophic factors for neurological diseases and tuning signaling pathways for heart failure, neurotrophic factors for neurological diseases, and tuning signaling pathways for heart failure and cancer.

[0054] Additional examples of gene addition strategies that the methods of the disclosure can target employ recombinant AAV delivery of genes encoding recombinant antibodies that can neutralize deadly viral infections. Such therapies would utilize intramuscular delivery and transform the transduced muscle cells into a biofactory to produce therapeutic antibodies that are secreted into the bloodstream. Such a strategy could target such infections and HIV, Hepatitis B or Hepatitis C.

[0055] Immunosuppressive agents such as the mTOR inhibitor, rapamycin, are increasingly used to prevent immune responses in gene therapy. These drugs block effector T cell and antibody responses and also promotes regulatory T cells. However, they also promote memory T cell responses. As a result, cessation of such administration of an immunosuppressive agent such as rapamycin may result in an immune response kicking in due to cessation of immune suppression. The present disclosure is based on the discovery that co-administration of rapamycin with an agent effective to suppress the functionality of interleukin- 15 (IL-15) receptors suppresses an immune response following cessation of rapamycin administration.

[0056] The disclosure also includes a method of treating a mammal, preferably, a human, undergoing gene therapy. The present disclosure provides a method for inhibiting an immune response in a subject undergoing AAV gene therapy comprising co-administering to the subject with an AAV gene therapy vector an effective amount of one or more of an IL-15 receptor blocker and one or more of immunosuppressive agents.

[0057] The IL-15 blocker and the immunosuppressive agent of certain embodiments of the present disclosure may be formulated with a pharmaceutical vehicle or diluent for oral, intravenous, subcutaneous, intranasal, intrabronchial or rectal administration. The pharmaceutical composition can be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration. Orally, the composition can be administered in the form of tablets, capsules, granules, powders and the like with at least one vehicle, e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc. The preparation may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol or the like and combination thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the composition. A preparation for parental administration includes sterilized water, suspension, emulsion, and suppositories. For the emulsifying agents, propylene glycol, polyethylene glycol, olive oil, ethyloleate, etc. may be used. For suppositories, traditional binders and carriers may include polyalkene glycol, triglyceride, witepsol, macrogol, tween 61, cocoa butter, glycerogelatin, etc. In addition, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like can be used as excipients.

[0058] The IL-15 blocker of the invention includes one or more of an antagonist of IL-15 receptors or antibody against IL-15 receptors. In any of the embodiments, the IL-15 blocker is one or more an agent effective to deactivate IL-15 receptors. In any of the embodiments of the invention the IL-15 blocker is administered prior to, concomitant with or following administration of one or more immunosuppressive agents or compounds.

[0059] The immunosuppressive agent of the current invention can be any immunosuppressive agent available clinically. More specifically, the immunosuppressive agent is an mTOR inhibitor. Preferably, the mTOR inhibitor is selected from one or more of rapamycin, temsirolimus, everolimus, ridaforolimus, tactolimus, or the like. More preferably, the mTOR inhibitor is rapamycin.

[0060] It is contemplated that the co-administration of each of the mTOR inhibitor and the IL- 15 blocker to the subject occurs within a suitable time prior to, during, or following gene therapy treatment. In some embodiments, the mTOR inhibitor and the IL-15 blocker are administered together. In yet other embodiments, mTOR inhibitor and the IL-15 blocker are administered separately.

[0061] The present invention provides a method of treating a mammal comprising: (a) administering a recombinant AAV virus vector to a mammal; and (b) co-administering each of an immunosuppressive agent and an IL-15 blocker to the mammal, each in an amount effective to have a therapeutic effect on said mammal. The therapeutic effect is preferably induction of a transient immune suppression to prevent an immune response that would impair the underlying AAV gene therapy.

[0062] Any of the agents of the invention, including the IL-15 blocker and the immunosuppressive agent can be administered by any route recognizable by the skilled clinical practitioner. Preferably, administration of the immunosuppressive agent occurs orally and the IL- 15 blocker is injected any one of intramuscularly, intraperitoneally or intravenously into the mammal. Alternatively, the immunosuppressive agent and the IL-15 blocker are administered any one or more of orally, intramuscularly, intraperitoneally or intravenously into the mammal.

[0063] It is contemplated that the IL-15 blocker is injected at a single site per dose or multiple sites per dose.

[0064] In any of the various embodiments of the current invention, the materials to accomplish any of the methods can be packed into a kit that includes all the necessary components to carry out said methods.

[0065] Further reference is made to the following experimental examples.

[0066] EXAMPLES

[0067] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are provided only as examples, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. [0068] EXAMPLE 1

[0069] Combination of Rapamycin and Anti-IL-15 to Preserve Transgene Expression and Allow for Re-administration in Hepatic AAV Gene Transfer

[0070] Hepatic gene transfer of codon-optimized human factor VIII (FVIII) with an adeno- associated virus serotype 8 (AAV8-co F8) in hemophilia A (HA) animal models can induce tolerance and durably replace FVIII. However, in some HA animal models tolerance is not always observed. For instance, it was observed that a sub-strain of BALB/c-HA reliably forms FVIII function-inhibiting antibodies (inhibitors) with this same approach.

[0071] The goal was to establish a regimen of transient immune suppression to prevent this response.

[0072] The inventors have previously shown that combining antigen, growth factor Flt3L, and the mTOR inhibitor rapamycin can induce tolerance to proteins via effector T cell deletion and regulatory T cell induction. Initial experiments were conducted to test the protocol during FVIII gene therapy in BALB/c-HA mice (1-2E11 vg/mouse vector dose, which sustains FVIII expression of -10-20% normal in B6/129-HA mice, a strain that does not develop inhibitors after hepatic gene transfer).

[0073] It was discovered that while inhibitor formation was merely delayed by several weeks in most of the immune-modulated BALB/c-HA mice compared to non-immune-suppressed controls, 25% never developed inhibitors and retained FVIII activity. A subsequent optimization experiment extended the protocol duration to ten weeks, and tested rapamycin alone or combined with Flt3L. Although no inhibitors formed, FVIII activity was mostly lost at later time points.

[0074] EXAMPLE 2

[0075] Combination of Rapamycin and Anti-IL-15 or IL-2/Anti-IL-2 Antibody Complexes or Anti-B-cell Activating Factor to Preserve Transgene Expression

[0076] Other experiments were conducted aimed to sustain FVIII activity levels through various alternative immunomodulators in combination with rapamycin. The immunomodulators included IL-2/anti-IL-2 antibody complexes, anti-B-cell activating factor, and anti-IL-15 (n=8- 10/group). While several regimens were partially successful, 8 weeks of rapamycin/anti-IL-15 combination achieved nearly undiminished FVIII levels throughout the 16-week experiment (mean=14.3%) (FIG. 1A-C). [0077] In comparison, FVIII levels in mice receiving only rapamycin steadily declined after it was stopped until near total loss. IL-15 is a cytokine critical to memory CD8+ T cells. Thus, it is possible that rapamycin-treated mice had a memory CD8+ T cell response that was blocked by anti-IL-15, thereby preserving therapeutic FVIII expression after rapamycin treatment was stopped. Interestingly, in rapamycin-treated mice, anti-AAV8 antibodies were only seen in vector- only mice (~10 pg/mL).

[0078] FIG. 1 A depicts the results showing that treatment with rapamycin or rapamycin + anti- IL-15 prevents antibody formation against human factor VIII (FVIII) in hepatic AAV8-hFVIII (AAV8-C0F8) gene transfer to hemophilia A mice (FIG. IB). However, anti-IL-15 was required to prevent loss of FVIII expression after rapamycin was discontinued (FIG. 1C).

[0079] EXAMPLE 3

[0080] Sustained expression of FVIII activity in hemophilia A mice after hepatic AAV gene transfer combined with immune modulation using rapamycin and anti-IL-15.

[0081] The results of Example 2 suggested that AAV re-administration might be possible. Thus, 17 weeks after the initial gene transfer, an identical dose of AAV8 vector expressing human FIX (n=7-10/group) was administered. FIG. 3A shows the injection protocols. Three weeks later, average FIX levels of 4.5 pg/mL and 2.7 pg/mL were measured for mice originally treated with rapamycin and rapamycin/anti-IL-15, respectively, versus 7.2 pg/mL in naive controls, indicating re-administration was successful. Without immune modulation with the first vector dose, mice failed to express FIX. All re-administered mice were immune competent and developed high levels of anti-AAV8 antibodies after the second gene transfer. Anti-IL-15 failed to suppress antibody formation in the absence of rapamycin.

[0082] FIG. 2A-C shows that no antibodies against AAV 8 capsid were detected 16 weeks after gene transfer in animals that had received treatment with either rapamycin or rapamycin + anti-IL- 15 (FIG. 2A). All animals received injections of a second AAV8 vector (same dose as original vector, 2xl0expl 1/mouse, n=7-9 per experimental group), this time, however, they expressed human factor IX (AAV8-hFIX). When tested 3 weeks later, those mice that received treatment with either rapamycin or rapamycin + anti-IL-15 showed similar FIX expression as previously naive control mice, while mice that had initially been injected with AAV8-FVIII vector without immune suppression failed to express FIX (FIG. 2B). After this second vector administration, which was done without immune suppression, animals of all experimental groups formed antibodies to AAV8 capsid (FIG. 2C). [0083] FIG. 3A shows that 6-8 week-old-old BALB/c-HA mice received intravenous AAV8- hFVIII (2xlOⁿ vg). Control mice (n=10) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=8) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). Note that control mice that did not receive immune suppression developed inhibitory antibodies against FVIII. However, mice that received rapamycin lost FVIII activity over time despite lack of antibody formation against FVIII. Only mice that received combination of rapamycin and anti-IL-15 showed sustained expression. 17 weeks after initial gene transfer, naive BALB/c-HA mice (n=8) and all mice previously administered AAV8-hFVIII, received AAV8-hFIX (2xlOⁿ vg). FIG. 3B depicts FVIII activity measured by chromogenic assay. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA at each time point, corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, pO.OOl; ****, pO.OOOl.

[0084] EXAMPLE 4

[0085] Eight weeks of rapamycin with AAV gene transfer prevents antibodies to FVIII and capsid, allowing AAV readministration.

[0086]

[0087] As in Example 3, 6-8 week-old BALB/c-HA mice received intravenous AAV8-hFVIII (2x10¹¹ vg). Control mice (n=10) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=8) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). 17 weeks after initial gene transfer, naive BALB/c-HA mice (n=8) and all surviving mice previously administered AAV8- hFVIII, received AAV8-hFIX (2x10¹¹ vg). At 16 weeks after gene transfer, as shown in FIG. 4 A, FVIII inhibitor antibodies were measured by Bethesda assay and as shown in FIG. 4B, ELISA measured a-AAV8 IgG2a antibodies. At 20 weeks after gene transfer, ELISA measured (FIG. 4C) circulating human clotting factor IX (hFIX) and (FIG. 3D) a-AAV8 IgG2a antibodies. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA, corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, pO.OOl; ****, pO.OOOl. [0088] EXAMPLE 5

[0089] Confirmation that anti-IL-15/rapamycin combination helps sustain FVIII activity in hemophilia A mice, while rapamycin treatment followed by CD8⁺ T cell depletion was less successful.

[0090] As in the previous examples, FIG 5A shows that 6-8 weeks-old BALB/c-HA mice received intravenous AAV8-hFVIII (2xlOⁿ vg). Control mice (n=6) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=21) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). After 8 weeks rapamycin-treated mice (n=8/21) received anti-CD8 (4 mg/kg) intravenously 2x/week for 8 weeks. FIG. 5B shows that FVIII activity was measured by chromogenic assay. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA at each time point and corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, pO.OOOl.

[0091] EXAMPLE 6

[0092] hFVIII gene copy numbers and mRNA transcripts are maintained regardless of FVIII activity, suggesting a translational shutdown that can be prevented by adding anti-IL-15 to rapamycin treatment.

[0093] Six-eight week-old BALB/c-HA mice received either received no treatment (naive; n=7) or intravenous AAV8-hFVIII (2x1011 vg). Control mice (n=6) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (6 mg/kg; n=21) or rapamycin plus anti-IL-15 (4 mg/kg; n=9). After 8 weeks, some rapamycin-treated mice received anti-CD8 (4 mg/kg; n=8) intravenously 2x/week for 8 weeks. At 16 weeks after gene transfer, (FIG. 6A) FVIII inhibitors were measured by Bethesda assay, (FIG. 6B) AAV copy number was measured by qPCR of DNA extracted from liver tissue, and (FIG. 6C) hFVIII and b-actin (reference gene) mRNA transcripts were measured by RT-qPCR of RNA extracted from liver tissue and fold change calculated by 2- AACt method. (FIG. 6D and 6E) A simple linear regression line was drawn comparing (FIG. 6D) AAV copy number and hFVIII mRNA and (FIG. 6E) hFVIII mRNA and FVIII activity. Error bars represent mean and standard error of the mean. Significance was determined by one-way ANOVA and corrected for multiple comparisons using Tukey post hoc test. *, p<0.05; **, p<0.01; ***, p O.OOl; ****, pO.OOOl. [0094] EXAMPLE 7— CHANGE FROM COLOR

[0095] Anti-IL-15 treatment protects AAV-FVIII transduced livers of hemophilia A mice from immune attack and preserves FVIII expression in hepatocytes.

[0096] Representative examples of liver sections with IHC staining for hFVIII (green), CD8 (red; white arrows), and nuclei (blue) are depicted in FIG. 7A-E. Six-eight week old BALB/c-HA mice received intravenous AAV8-hFVIII (2xlOⁿ vg). Control mice (n=6) received vector only, while other mice also received immune modulation intravenously 2x/week for 8 weeks. Immune modulation regimens consisted of either rapamycin (4 mg/kg), or rapamycin/anti-IL-15 (4 mg/kg). After 8 weeks, some rapamycin-treated mice received anti-CD8 (4 mg/kg) intravenously 2x/week for 8 weeks. (FIG. 7A) naive mice or 16 weeks after gene transfer: (FIG. 7B) AAV only or with regimens of (FIG. 7C and FIG. 7D) rapamycin, (D) rapamycin/anti-CD8, or (FIG. 7E) rapamycin/anti-IL- 15.

[0097] In summary, transient rapamycin treatment with hepatic AAV gene transfer prevents antibody formation against transgene product and vector.

[0098] As will be appreciated from the descriptions herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, without limitation, the aspects and embodiments listed below:

[0099] A method is presented for preserving AAV gene therapy through transient immune suppression, comprising co-administering to a subject undergoing AAV gene therapy an effective amount of an immunosuppressive agent and an IL-15 blocker, the combination of an immunosuppressive agent and IL-15 blocker being effective to preserve therapeutic expression of a transgene delivered by said AAV gene therapy.

[00100] A method in accordance with any other embodiment disclosed herein, wherein the co administering an effective amount of in immunosuppressive and an interleukin- 15 blocker is effective to suppress a deleterious immune response against the AAV gene therapy.

[00101] A method in accordance with any other embodiment disclosed herein, wherein co administering an effective amount of an immunosuppressive agent and an interleukin- 15 blocker is effective to suppress a memory CD8⁺ T cell response.

[00102] A method in accordance with any other embodiment disclosed herein, wherein the AAV vector is a liver-targeting AAV, an AAV mediated liver gene therapy or a liver gene transfer vector. [00103] A method in accordance with any of the embodiments disclosed herein, wherein the IL- 15 blocker includes one or more of an antagonist of IL-15 receptors, an antibody against IL-15 receptors or one or more an agents effective to deactivate IL-15 receptors.

[00104] A method in accordance with any of the embodiments disclosed herein, wherein the IL- 15 blocker is administered prior to, concomitant with or following administration of one or more immunosuppressive agents or compounds.

[00105] A method in accordance with any of the embodiments disclosed herein, wherein the immunosuppressive agent is an mTOR inhibitor and where the mTOR inhibitor is selected from one or more of rapamycin, temsirolimus, everolimus, ridaforolimus, tactolimus, or the like. [00106] A method in accordance with any of the embodiments disclosed herein, wherein the mTOR inhibitor is preferably rapamycin.

[00107] A method in accordance with any of the embodiments disclosed herein, wherein the co administration of each of the mTOR inhibitor and the IL-15 blocker to the subject occurs within a suitable time prior to, during, or following gene therapy treatment. In some methods, the mTOR inhibitor and the IL-15 blocker are administered together or alternatively, the mTOR inhibitor and the IL-15 blocker are administered separately.

[00108] A method is presented of treating a mammal comprising: (a) administering a recombinant AAV virus vector to a mammal; and (b) co-administering each of an immunosuppressive agent and an IL-15 blocker to the mammal, each in an amount effective to have a therapeutic effect on said mammal.

[00109] A method in accordance with any of the embodiments disclosed herein, wherein the method elicits a therapeutic effect where the therapeutic effect is to induce a transient immune suppression to prevent an immune response that would impair the underlying AAV gene therapy. [00110] A method in accordance with any of the embodiments disclosed herein, wherein the mammal is a patient in need of gene therapy treatment.

[00111] A method in accordance with any of the embodiments disclosed herein, wherein the administration of the immunosuppressive agent occurs orally and the IL-15 blocker is injected any one of intramuscularly, intraperitoneally or intravenously into the mammal. Alternatively, the immunosuppressive agent and the IL-15 blocker are administered any one or more of orally, intramuscularly, intraperitoneally or intravenously into the mammal.

[00112] A method in accordance with any of the embodiments disclosed herein, wherein the IL- 15 blocker is injected at a single site per dose or multiple sites per dose. [00113] A method in accordance with any of the embodiments disclosed herein, wherein all of the materials can be packaged into a kit containing all of the necessary components to carry out the claimed methods.

[00114] While embodiments of the present disclosure have been described herein, it is to be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for preserving adeno associated virus (AAV) gene therapy through transient immune suppression, comprising co-administering to a subject undergoing AAV gene therapy an effective amount of an immunosuppressive agent and an interleukin- 15 (IL-15) blocker, the combination of the immunosuppressive agent and IL-15 blocker being effective to preserve therapeutic expression of a transgene delivered by said AAV gene therapy.

2. The method of claim 1, wherein said co-administering is effective to suppress a deleterious immune response against the AAV gene therapy.

3. The method of claim 1, wherein said co-administering is effective to suppress a memory CD8⁺ T cell response.

4. The method of claim 1, wherein the AAV vector comprises one or more of a liver- targeting AAV, AAV mediated liver gene therapy and a liver gene transfer vector.

5. The method of claim 1, wherein the IL-15 blocker comprises an antagonist of IL-

15 receptors.

6. The method of claim 1, wherein the IL-15 blocker comprises an agent effective to deactivate IL-15 receptors.

7. The method of claim 1, wherein the immunosuppressive agent is an mTOR inhibitor.

8. The method of claim 7, wherein the mTOR inhibitor is one or more of inhibitors selected from rapamycin, temsirolimus, everolimus, ridaforolimus, and tacrolimus.

9. The method of claim 8, wherein the mTOR inhibitor is rapamycin.

10. The method of claim 1, wherein the co-administering comprises administering each of immunosuppressive agent and the IL-15 blocker to the subject within a suitable time prior to, during or following a gene therapy treatment.

11. A method of treating a mammal comprising: (a) administering a recombinant adeno associated virus (AAV) vector to a mammal; and (b) co-administering each of an immunosuppressive agent and an IL-15 blocker to the mammal, each in an amount effective to have a therapeutic effect on said mammal.

12. The method of claim 11, wherein the therapeutic effect is the suppression of a deleterious immune response against the AAV gene therapy.

13. The method of claim 11, wherein the mammal is a patient in need of gene therapy treatment.

14. The method of claim 11, wherein the co-administering comprises orally administering the immunosuppressive agent and injecting the IL-15 blocker into the muscle tissue of said mammal.

15. The method of claim 11, wherein the co-administering comprises orally administering the immunosuppressive agent and injecting the IL-15 blocker intraperitoneally into said mammal.

16. The method of claim 11, wherein the IL-15 blocker is injected at a single site per dose.

17. The method of claim 11, wherein the IL-15 blocker is injected at multiple sites.

18. The method of claim 11, wherein the IL-15 blocker and the immunosuppressive agent are administered at the same time.

19. The method of any of claims 11-18, the immunosuppressive agent is one or more of an mTOR inhibitor selected from rapamycin, temsirolimus, everolimus, ridaforolimus, and tacrolimus.

20. A method of treating a mammal comprising: (a) administering a recombinant adeno associated virus (AAV) vector to a mammal; and (b) co-administering each of rapamycin and an IL-15 blocker to the mammal, each in an amount effective to have a therapeutic effect on said mammal.